Influenza A viruses (IAV) pose a major public health threat through both seasonal epidemics and sporadic pandemics. The segmented nature of the viral genome promotes reassortment, a process where the genetic material between viruses is exchanged in a co-infected cell. In nature, reassortment leads to increased viral diversity and emergence of pandemic influenza viruses. For example, the 2009 influenza H1N1 (?swine flu?) pandemic virus, emerged from reassortment of two circulating swine viruses. Prediction of future pandemic influenza viruses from circulating zoonotic virus populations is difficult because very little is known about the mechanism of reassortment within a single co-infected cell. To accurately define the process of reassortment, we must first understand the dynamics of intracellular viral RNA (vRNA) assembly. Influenza vRNA replicates in the nucleus and is transported to the plasma membrane for packaging, which requires one copy of all eight segments to assemble within a single virion to produce a fully infectious virus. In this proposal, we will build upon our previous data on influenza assembly and define 1) the assembly dynamics in physiologically relevant human and swine cell types, 2) the cellular proteins modulating vRNA transport, and 3) the location of reassortment within a co-infected cell. Our central hypothesis is that vRNA assembly occurs in a cell-type specific manner that correlates with IAV reassortment in different host species. The Specific Aims of this application will use a variety of sophisticated microscopy tools, including live cell imaging with a custom light-sheet microscope, to determine the assembly mechanism in various cell culture models. Aim 1 will utilize multicolor fluorescent in situ hybridization and live cell imaging techniques to explore the dynamics of influenza vRNA assembly in human and swine differentiated airway epithelial cells. Aim 2 will uncover the identity and roles of cellular cytoskeletal proteins and membranous organelles utilized during influenza vRNA assembly using biochemical approaches like proximity-dependent biotinylation. Aim 3 will combine imaging and genomic approaches to characterize the cellular location of vRNA intermingling during co-infection with two heterologous viruses in differentiated airway epithelial cells. The proposed work will address many outstanding questions in influenza biology regarding reassortment that have remained unanswered due to a lack of tools to track vRNA movement in live cells during a productive infection. In addition, these studies will identify novel host factors involved in vRNA packaging that can be pursued as potential therapeutic targets.